Conjugated linoleic acid protects against age-associated bone loss in C57BL/6 female mice

Osteoporosis is one of the major causes of morbidity in the elderly. Inflammation exerts a significant influence on bone turnover, inducing the chronic form of osteoporosis. Dietary nutrition has the capacity to modulate inflammatory response. Therefore, nutritional strategies and lifestyle changes may prevent age-related osteoporosis, thereby improving the quality of life of the elderly population. Conjugated linoleic acid (CLA) has been shown to positively influence calcium and bone metabolism. Hence, this study was undertaken to examine the effect of CLA on bone mineral density (BMD) in middle-aged C57BL/6 female mice. After 10 weeks on diet, CLA-fed mice (14 months) maintained a higher BMD in different bone regions than corn oil (CO)-fed mice. The increased BMD was accompanied by a decreased activity of proinflammatory cytokines (such as tumor necrosis factor alpha, interleukin-6 and the receptor activator of NF-kappaB ligand) and decreased osteoclast function. Furthermore, a significant decrease in fat mass and an increase in muscle mass were also observed in CLA-fed mice compared to CO-fed mice. In conclusion, these findings suggest that CLA may prevent the loss of bone and muscle mass by modulating markers of inflammation and osteoclastogenic factors.     

Quantitative trait loci for bone density in C57BL/6J and CAST/EiJ inbred mice

Genetic analyses for loci regulating bone mineral density have been conducted in a cohort of F₂ mice derived from intercross matings of (C57BL/6J × CAST/EiJ)F₁ parents. Femurs were isolated from 714 4-month-old females when peak adult bone density had been achieved. Bone mineral density (BMD) data were obtained by peripheral quantitative computed tomography (pQCT), and genotype data were obtained by Polymerase Chain Reaction (PCR) assays for polymorphic markers carried in genomic DNA of each mouse. Genome-wide scans for co-segregation of genetic marker data with high or low BMD revealed loci on eight different chromosomes, four of which (Chrs 1, 5, 13, and 15) achieved conservative statistical criteria for suggestive, significant, or highly significant linkage with BMD. These four quantitative trait loci (QTLs) were confirmed by a linear regression model developed to describe the main effects; none of the loci exhibited significant interaction effects by ANOVA. The four QTLs have been named Bmd1 (Chr 1), Bmd2 (Chr 5), Bmd3 (Chr 13), and Bmd4 (Chr 15). Additive effects were observed for Bmd1, recessive for Bmd3, and dominant effects for Bmd2 and Bmd4. The current large size of the QTL regions (6→31 cM) renders premature any discussion of candidate genes at this time. Fine mapping of these QTLs is in progress to refine their genetic positions and to evaluate human homologies. Received: 5 May 1999 / Accepted: 22 June 1999     

Long-term isolated housing causes anxiety in C57BL/6J female mice

Behaviour of female C57BL/6J strain mice was studied in the elevated plus-maze and Porsolt's tests after either long-lasting individual housing or keeping with daily shifting group-housed females (social instability). After 2-3 months, an increased level of anxiety in the individually housed females was revealed in the elevated plus-maze. However, in 3 months the least passive behaviour in the Porsolt's was in the individually housed females. No changes were found in behaviour of females individually housed at 3 weeks of age for 4 months. Also, females with preliminary social contacts with males and following individual housing for one month had not any abnormalities in the used behavioural tests. Social instability conditions did not significantly influence the females' plus-maze behaviour, but decreased the passive behaviour in the Porsolt's test.     

Effect of dietary restriction on estrous cyclicity and follicular reserves in aging C57BL/6J mice

Restricting the food intake of female mice by alternating days of feeding and fasting delayed the age-related loss of estrous cycling potential and retarded the rate of follicular depletion, as determined after reinstatement of ad libitum (AL) feeding. During the period of food restriction (FR; 3.5-10.5 mo), food intake and body weight were about 80% of AL values. Mice were acyclic and predominantly in a state of diestrus during FR, but after reinstatement of an AL diet at 10.5 mo all FR mice resumed cycling regularly. By contrast, 80% of AL controls had become acyclic by this age, and the cycles of the remaining mice were significantly longer than those of the reinstated FR mice. Follicular reserves of 12.5-mo-old FR mice were twice those of age-matched AL controls. Cycling performance of reinstated FR mice, measured by cycle length and the proportion of mice still cycling, was equivalent to that of AL mice when the latter were 2-5 mo younger. Ovarian age, measured by the size of the follicular reserve, was similarly retarded in FR mice. Based on these data and previous evidence that follicular depletion plays a major role in the cessation of cyclicity in this strain, we hypothesize that the delayed loss of estrous cyclicity in aging FR mice is mediated at least in part by the retarding effect of dietary restriction on the rate of follicular depletion.     

Embryonic stem cells derived from C57BL/6J and C57BL/6N mice

Mouse embryonic stem (ES) cells with the C57BL/6 genetic background allow the generation of knockout mice without the need to backcross to C57BL/6. However, C57BL/6 ES cells whose pluripotency after homologous recombination has been confirmed are not yet available from public cell banks. To facilitate the use of ES cells derived from C57BL/6 sublines in both biologic and medical research, we demonstrated that the use of knockout serum replacement as a medium supplement and 8-cell blastomeres as recipient embryos allowed establishment of ES cells and production of germline chimeric mice, respectively. Under effective conditions, a large number of ES cell lines were established from C57BL/6J and C57BL/6N blastocysts. The majority of ES cells in many cell lines obtained from both strains showed a normal chromosome number. Germline chimeric mice were generated from C57BL/6J and C57BL/6N ES cells. Finally, the ES cell line B6J-S1UTR, derived from C57BL/6J, was used for successful production of gene knockout mice. C57BL/6J ES (B6J-S1UTR and B6J-23UTR) and C57BL/6N ES (B6N-22UTR) cells are available from the cell bank of the BioResource Center at RIKEN Tsukuba Institute (http://www.brc.riken.jp/lab/cell/english/).     

Differences in anxiety-related behavior between apolipoprotein E-deficient C57BL/6 and wild type C57BL/6 mice

The influence of ApoE gene deletion on the anxiety state has not been previously investigated. The elevated plus maze was used in this study to determine differences in anxiety-related behavior between apoE-deficient and wild type C57BL/6 mice. The apoE-deficient mice demonstrated less anxiety on the elevated plus maze by spending more time in the open arms of the elevated plus maze compared to wild type mice (p<0.001). Additionally, female apoE-deficient mice visited the open arm of the maze more often than their apoE-deficient male counterpart (p<0.05). The anxiety state and/or sex are possible variables to be considered when designing physiological and/or behavioral studies involving mice that are apoE-deficient.     

Effects of housing density and cage floor space on C57BL/6J mice

The Guide for the Care and Use of Laboratory Animals (the Guide) is widely accepted as the housing standard by most Institutional Animal Care and Use Committees. The recommendations are based on best professional judgment rather than experimental data. Current efforts are directed toward replacing these guidelines with data-driven, species-appropriate standards. Our studies were undertaken to determine the optimum housing density for C57BL/6J mice, the most commonly used inbred mouse strain. Four-week-old mice were housed for 8 weeks at four densities (the recommended approximately 12 in2 [ca. 77.4 cm2]/mouse down to 5.6 in2 [ca. 36.1 cm2]/mouse) in three cage types with various amounts of floor space. Housing density did not affect a variety of physiologic parameters but did affect certain micro-environmental parameters, although these remained within accepted ranges. A second study was undertaken housing C57BL/6J mice with as little as 3.2 in2/mouse (ca. 20.6 cm2). The major effect was elevated ammonia concentrations that exceeded limits acceptable in the workplace at increased housing densities; however, the nasal passages and eyeballs of the mice remained microscopically normal. On the basis of these results, we conclude that C57BL/6J mice as large as 29 g may be housed with 5.6 in2 of floor space per mouse. This area is approximately half the floor space recommended in the Guide. The role of the Guide is to ensure that laboratory animals are well treated and housed in a species-appropriate manner. Our data suggest that current policies could be altered in order to provide the optimal habitation conditions matched to this species' social needs.     

Estrogen and progesterone affect responses to malaria infection in female C57BL/6 mice

Background: Previous data from our laboratory suggest that gonadally intact C57BL/6 male mice are more likely than their female counterparts to die from Plasmodium chabaudi infection, to recover more slowly from weight loss and hematocrit loss, and to have reduced interferon-γ (IFN-γ) and interleukin-10 (IL-10) responses. Removal of the ovaries, and hence, the primary production of sex steroids in females, reverses these differences.Objective: We hypothesized that sex differences in response to P chabaudi may be mediated by differential synthesis of IFN-γ and IL-10 that is influenced by estrogen, progesterone, or both.Methods: C57BL/6 female mice (n = 200; n = 10/time point/treatment/experiment) were ovariectomized and implanted with a 21-day controlled-release pellet containing either 0.1 mg of 17β-estradiol (E₂), 10 mg of progesterone (P₄), 0.1 mg of E₂ plus 10 mg of P₄, or cholesterol (placebo). Females were inoculated with 10⁶P chabaudi-infected erythrocytes. Body mass, body temperature, hematocrit, parasitemia, cytokine production, and antibody responses were monitored 0, 3, 5, 7, 10, 14, and 21 days postinoculation.Results: Administration of E₂, either alone or in combination with P₄, mitigated infection-induced weight loss, hematocrit loss, and hypothermia, compared with females receiving placebo pellets (P < 0.05 in each case). Hormone treatment did not affect levels of parasitemia. Females administered E₂ alone or in combination with P₄ produced 4 to 7 times higher IFN-γ and IL-10 during peak parasitemia than did females implanted with pellets containing either P₄ alone or placebo (P < 0.05 in each case). Exposure to E₂, either alone or in combination with P₄, increased anti-P chabaudi immunoglobulin G (IgG1) responses and the ratio of IgG1 to IgG2c (P < 0.05 in each case).Conclusion: This animal study suggests that physiological levels of estrogen, rather than progesterone, enhance immunity and, possibly, protect females from disease symptoms during malaria infection.     

Delayed matching-to-position performance in C57BL/6N mice

Delayed matching-to-sample is one of the most frequently employed behavioral tasks for assessing spatial working memory in animals. Although the advantages of the task have been widely acknowledged and it is used in the study of a variety of species, its application to mice has been rare. In the present study, we reported the efficacy of a delayed matching-to-position task in C57BL mice lever-pressing in an operant-conditioning chamber. Each trial started with the press of a back lever, followed by the presentation of either a left or right front lever. When the ratio requirement for presses to the front lever (sample) was met, a delay interval started. Delay interval continued until the mice made the first response after the elapse of the programmed delay interval. This was followed by the presentation of a choice of left or right front levers. The choice of the same front lever as the sample was reinforced, whereas the other was not. The proportion of correct choices showed a delay-dependent decrement. A higher ratio of response requirement to the sample resulted in increased accuracy, but the duration of the intertrial interval had no effect. Preceding trials also influenced response accuracy, indicating proactive interference. Overall, the results replicated the effects of parametric manipulations reported in other species, and thus, our findings validate the efficacy of the task for assessing spatial working memory in laboratory mice.     

Tumours of the bone in C57BL/10J mice

Tumours of the bone were found in 17 (12 females and 5 males) of 6768 (0.25%) C57BL/10J mice (3384 males and 3384 females) pooled from 14 oncogenic studies carried out from 1973 to 1987. Tumours of bone were found predominantly in female mice and were age related. The tumours were classified histologically as osteosarcoma (5 osteoblastic, 6 mixed type and 1 fibroblastic), chondroma (2), chondromyxoid fibroma (1), chondrosarcoma (1) and periosteal fibroma (1).     

Neurogenesis of the cholinergic medial septum in female and male C57BL/6J mice

Sex differences exist in the structure and function of the cholinergic septo-hippocampal system throughout the lifespan of mammals. How and when these sex differences originate is unclear. Because estrogen modulates sexual differentiation of several brain regions during development and influences neurogenesis in adult mammals, we hypothesized that sexual dimorphism of the cholinergic septo-hippocampal system would extend to its neurogenesis. A birthdating agent 5'-bromo-2'-deoxyuridine (BrdU) was injected into pregnant dams on one of eight gestational days, ranging from embryonic day (E)10 to E17. The offspring were euthanized at 2 months of age, and brains were processed for BrdU and choline acetyltransferase (ChAT) immunoreactivity to label cholinergic neurons that became postmitotic on a given embryonic day and survived to adulthood. Unbiased stereology was used to compare the number of double-labeled neurons in the medial septum (MS) of female and male offspring. Cholinergic neurons in the MS were generated primarily between E11 and E14, similar to other published reports. We found sex differences in the pattern of peak neurogenesis but not in the length of neurogenesis, or in total number of neurons generated in the MS. Additionally, in adult female and male mice, we estimated the total number of cholinergic neurons using unbiased stereology and found no sex differences in the number of cholinergic neurons or in the volume of the MS in adulthood. These results suggest that sex differences noted in the function of the postnatal cholinergic septo-hippocampal system may originate from its neurogenesis.     

Prepubertal OVX increases IGF-I expression and bone accretion in C57BL/6J mice

It is generally well accepted that the pubertal surge in estrogen is responsible for the rapid bone accretion that occurs during puberty and that this effect is mediated by an estrogen-induced increase in growth hormone (GH)/insulin-like growth factor (IGF) action. To test the cause and effect relationship between estrogen and GH/IGF, we evaluated the consequence of ovariectomy (OVX) in prepubertal mice (C57BL/6J mice at 3 wk of age) on skeletal changes and the GH/IGF axis during puberty. Contrary to our expectations, OVX increased body weight (12-18%), bone mineral content (11%), bone length (4%), bone size (3%), and serum, liver, and bone IGF-I (30-50%) and decreased total body fat (18%) at 3 wk postsurgery. To determine whether estrogen is the key ovarian factor responsible for these changes, we performed a second experiment in which OVX mice were treated with placebo or estrogen implants. In addition to observing similar results compared with our first experiment, estrogen treatment partially rescued the increased body weight and bone size and completely rescued body fat and IGF-I levels. The increased bone accretion in OVX mice was due to increased bone formation rate (as determined by bone histomorphometry) and increased serum procollagen peptide. In conclusion, contrary to the known estrogen effect as an initiator of GH/IGF surge and thereby pubertal growth spurt, our findings demonstrate that loss of estrogen and/or other hormones during the prepubertal growth period effect leads to an increase in IGF-I production and bone accretion in mice.     

Age-related fibrillar deposits in brains of C57BL/6 mice

The present article reviews findings regarding the age-related occurrence of clusters of unusual granules in the brains of C57BL/6 (B6) mice and discusses the potential relevance of this phenomenon as a model of specific aspects of brain aging in humans. The granules occur predominatly in the hippocampus of B6 mice and represent aggregations of fibrillar material that are mostly associated with astrocytes. The deposits become evident at about 4 to 6 mo of age, and increase markedly in both number and size thereafter. Similar structures have been observed in adult senescence accelerated mice (SAM) and have been noted, although very rarely, in older mice from other strains. The deposits appear to manifest dominant genetic heritability. Heparan sulfate proteoglycan and laminin or related molecules have been identified as components of the granular material. Although the deposits do not represent senile plaques with β-amyloid deposition, they might mimic the deposition of extracellular matrix molecules that is thought to be an early event in amyloidogenesis in the aged brain and in Alzheimer's disease.     

Self-mutilation of the penis in C57BL/6N mice

A major cause of male reproductive failure in a C57BL/6N mouse production colony is self-inflicted mutilation of the penis. The extent of the damage ranged from loss of the distal end to loss of the entire penis. From January 1974 to August 1976, 645 adult male mice with mutilated penis were removed from this colony--where the monthly census was 9500 mice, mated 1 male to 4 females using a continuous mating system. On necropsy, it was observed that the substance blocking the urethra in the penile stump resulted in urine retention, grossly distending the urinary bladder. Proteus mirabilis and other bacteria isolated from the urethral plugs were considered secondary invaders.     

Protective effect of bone marrow and spleen suspensions on radiation-induced leukemogenesis in C57BL/6 mice

Summary The present experiments are an attempt to precise the type and localization of the cells involved in the protective effect of hemopoietic suspensions against the radiation-induced thymic lymphosarcoma (TLS) of C57BL/6 mice. Inocula containing variable numbers of BM or spleen CFUs from 60-day-old and 360-day-old donors were tested. According to their origin, the suspensions differed with respect to the CFU replication rate, the CFU ability to differentiate towards the T lineage and the content of the suspensions in thymic precursors. Two levels of inhibition were observed: BM suspensions from 60-day-old donors containing 1,500 CFUs had the best protective effect: 14.5% of TLS; 1,500 CFUs from 360-day-old donors were slightly but not significantly less efficient (28.5%). The second level of inhibition (36–46% of TLS) was obtained with all the following inocula:a) 1,200 and 300 spleen CFUs or 300 and 95 BM CFUs from 60-day-old donors,b) 1,500 spleen CFUs from aged donors. Seventy-six spleen CFUs from 60-day-old donors, 120 BM or 175 spleen CFUs from aged donors had no effect. These results suggest that in addition to the high replication rate of the BM CFUs as compared with spleen CFUs, cells endowed with an optimal protective effect are present in BM suspensions and are either absent or present in very small amount in spleen suspensions. These cells which induce an early repopulation of the thymus might correspond to thymic precursors.